The invention relates to a surface plasmon resonance sensor for the simultaneous measurement of a plurality of samples present in fluid form that permits a fast sample measurement within the frame of various application purposes. In particular, the sensor according to the present invention is utilized in parallel or serial measurement of samples, which are contained in micro-titer plates.
Due to the more and more expedited automation in the field of search for effective substances, the question of miniaturization and parallelizing finds an increasing interest. The miniaturization of sample receptacles and apparatus for synthesis and the parallelizing of the sequence of procedures leads to a plurality of substances to be tested which are of less and less volume. Thus, when implementing novel detection systems and sensor systems it is necessary to embody the detection systems and sensor systems in such a manner to enable a simultaneous and parallel execution of a plurality of measurements, respectively, a subsequent measurement of a great number of samples within a shortest time, wherein the amount of substances required is minimized. Thereby, the increase of the degree of automation plays an important role.
Background of the invention is the need to provide also the sensors used in measurements in a parallel and miniaturized design so that the measurement of a plurality of samples can be carried out in the shortest possible time and with a minimum of sample volume and expenditures and, thus, to increase the throughput of substances to be identified.
There is known a very sensitive method for specifying the characteristics of boundary faces that, in the references, is referred to as surface plasmon resonance spectroscopy, generally designated as SPR (surface plasmon resonance). This method is based upon the optical excitation of surface plasmons in thin metal layers. According to the state of art, this method has been described, inter alia, in detail by Striebel, Ch.; Brecht, A.; Gauglitz, G. in Biosensors and Bioelectronics 9 (1994), 139-146. The resonance conditions for the excitation of surface plasmons strongly depend on the optical properties of the dielectrics surrounding the metal layer. According to the prior art it is generally feasible with high precision to determine the refractive index and the layer thickness of thin dielectric layers. SPR-spectroscopy finds an increasing use, for example, in the biochemical analysis, since it permits a direct investigation of the interactions between the bio-molecules (for example, antibody/antigen reactions). To this end a reactant (ligand) is immobilized on the metal surface, the other reactant (analyt) is passed over the surface in solution. The interaction can be directly detected as an increase in layer thickness via the refractive index change.
Conventional SPR-sensors (refer to product specification of the firm Biacore AB, Rapsgatan 7, S-75450 Uppsala, Sweden 1996) employ a prism which supports a thin metal layer. The sample to be measured is brought into contact to the metal, respectively, to the modified metal surface, and the SPR-reflection spectrum of the sample is measured by coupling-in light and measuring the intensity of the reflected light as a function of the angle of incidence or of the wavelength.
Recent methods and devices (WO 94/16312) employ fiber-optical elements for setting up SPR-sensors. Thereby commercially available light conducting fibers are used, having a diameter of from 1 xcexcm to 2000 xcexcm. The fibers or other defined portions thereof are dismantled, that is, the covering which consists of a wave guide cover and a buffer layer, are removed mechanically or chemically or thermally. Subsequently, the fibers are radially or partially radially provided with a metal layer and, when employing fiber-optical sensor operating as an end-reflector, the leading face of the fiber is additionally coated. Thereby, there are very high standards required from the radial coating as to the homogeneity of the layer thickness that can only be realized technologically under high expenditures.
A further disadvantage when using light conducting fibers lies in the reduced chance for a parallelizing, since single light conducting fibers have always to be arranged manually to obtain an array.
It is an object of the present invention to provide an SPR-sensor for the simultaneously measurement a plurality of samples present in fluid form that can be arranged to a preselectable array, wherein the SPR-sensors will be manufactured by way of a uniform technology and at less expenditures than involved by those of the prior art.
The object is realized by features of the first patent claim. Preferable embodiments are subject matter of the dependent claims.
The object of the invention is realized by planar waveguides, each of which being provided with at least one SPR-sensor area. SPR-sensor according to the invention can be arranged in parallel and can simultaneously be brought into contact with a great number of samples (greater 100).
The planar waveguides used thereby conduct the excitation light to the sensor area that operates on the measuring principle of the surface plasmon resonance in order to measure a solution brought into contact to the sensor. Thereby exactly one sample is brought into contact with one respective sensor area so that it is feasible to determine n-different samples with one SPR-waveguide array constituted of n-waveguides. One SPR-waveguide array will be manufactured by way of utilizing technologies from the semiconductor production and from the integrated optics to provide in parallel a great number of sensors and to arrange the same at a defined distance to one another.
According to the invention it is also feasible to integrate the SPR-waveguide arrays in sample receptacles, for example, in micro-titer plates. Thereby, the SPR-waveguide arrays are adapted to match with already existing sizes of micro-titer plates (96, 386, 1536 etc.), but also to novel formats or to such ones departing from the already existing formats.
Planar waveguides are increasingly taken notice of in research and development in the field of integrated optics. A light conducting layer is deposited level to a support material when manufacturing planar waveguides. The refractive index of the support material or a layer adapted thereupon to that purpose has to be lower than the refractive index of the waveguiding layer to ensure that the light in the waveguide is guided substantially without any loss. Such planar waveguides are produced by use of known technologies of the semiconductor techniques and integrated optics such as, for example, CVD-processes, sputtering, electron beam vaporization, centrifugation or various replication techniques. It is also feasible to manufacture minutely structurized waveguides and wave branching elements under use of known micro-technological methods of structurizing. Thereby and by use of diverse structurizing methods, waveguides can be produced having a thickness in a range of from a few xcexc-meters up to some 100 xcexcm and widths up to some 1000 xcexcm. The coating of defined waveguide sections with a layer capable of SPR can also be carried out in parallel with a few steps by known technologies.
An SPR-sensor according to the present invention is comprised of a plurality of planar stripe-shaped light wave guides that are provided, between respective two leading faces, with at least one two-dimensional measuring area. These measuring areas are coated with a planar metal layer capable of SPR that is in direct contact with both, the waveguiding material and the sample to be determined.
The excitation light enters the light wave guide via known coupling mechanisms. There the light propagates in and along the waveguide and is guided to the sensor area. In the sensor area the light guided in the light wave guide is affected by excitation of the surface plasmons.
In the further course, the modified light is either coupled-off from the light wave guide by way of the known coupling principles directly after passing the sensor area and is passed on to further processing; or it is back-reflected in itself in the light wave guide by means of a mirror coating deposited on the leading face and is coupled-off again via the same coupling mechanism by which the light entered into the light wave guide, and is thus provided for further processing.
When the light is coupled-in and coupled-out at one and the same side of the light wave guide and the reflection of the radiation takes place at the other end, then planar SPR-waveguides on the basis of end-reflection are concerned. When the coupled-in light leaves the waveguide at the second side of the waveguide then one speaks of waveguide sensors based on inline-transmission.